The broad, long-term objective of the proposed research is to determine the mechanisms of von Willebrand Factor (VWF) function. We will study the binding of the VWF A1 domain to platelet glycoprotein Ib? (GPIb?). We hypothesize, based on crystal structures and single molecule measurements of A1-GPIb? unbinding, that force can regulate the binding of A1 to GPIb? by switching the A1-GPIB? bond into a high affinity state. The high affinity state potentially mimics the state of the A1 domain induced by ristocetin, an antibiotic that promotes VWF mediated platelet agglutination. We propose to measure the on- and off-rates of A1-GPIb? binding to gain a mechanistic understanding of its regulation by force and ristocetin. Specific Aim 1: Characterize the affect of Ristocetin on A1-GPIb? bond kinetics. We will generate A1 domain constructs with and without ristocetin binding epitopes. A1-GPIb? binding kinetics and affinity will be measured in the presence and absence of ristocetin using Surface Plasmon Resonance (SPR). These measurements will quantitatively determine how ristocetin enhances A1-GPIb? binding, which is important for the diagnosis and treatment of Von Willebrand Disease (VWD). This aim will provide novel training for the fellow in recombinant protein expression in mammalian cells, protein purification and immobilization, and SPR. Specific Aim 2: Determine the force-dependent on-rate of A1-GPIb?. We have designed a Receptor and Ligand in a Single Molecule (ReaLiSM) containing the VWF A1 domain linked by a polypeptide chain to GPIb?. ReaLiSM allows repeated measurements of unbinding and binding using laser tweezers. We will measure the Wild Type, A1-GPIb? on-rate as a function of force +/- ristocetin using ReaLiSM. The results will determine whether flexing between a high and low affinity state can enhance A1-GPIb? binding. The fellow will receive training in all experimental and computational aspects of single molecule force spectroscopy. Specific Aim 3: Characterize the bond kinetics of gain-of-function (GOF) A1 and GPIb? mutations to understand the basis of Von Willebrand Disease (VWD). Using laser tweezers and SPR, we will measure the binding kinetics and mechanical properties of A1- GPIb? containing VWD-2B and platelet-VWD (PT-VWD) mutations. The results will quantify the mechanical effects of these VWD mutations on binding kinetics, and will determine whether a combination of mutations can shift the bond into a single, high affinity state.